Bicyclic peptide oligonucleotide conjugates

ABSTRACT

Provided herein are oligonucleotides, bicyclic peptides, and peptide-oligonucleotide-conjugates. Also provided herein are methods of treating a muscle disease, a viral infection, or a bacterial infection in a subject in need thereof, comprising administering to the subject oligonucleotides, peptides, and peptide-oligonucleotide-conjugates described herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser. No. 62/573,473, filed Oct. 17, 2017, which is incorporated herein by reference in its entirety.

BACKGROUND

Antisense technology provides a means for modulating the expression of one or more specific gene products, including alternative splice products, and is uniquely useful in a number of therapeutic, diagnostic, and research applications. The principle behind antisense technology is that an antisense compound, e.g., an oligonucleotide, which hybridizes to a target nucleic acid, modulates gene expression activities such as transcription, splicing or translation through any one of a number of antisense mechanisms. The sequence specificity of antisense compounds makes them attractive as tools for target validation and gene functionalization, as well as therapeutics to selectively modulate the expression of genes involved in disease.

Although significant progress has been made in the field of antisense technology, there remains a need in the art for oligonucleotides and peptide-oligonucleotide-conjugates with improved antisense or antigene performance. Such improved antisense or antigene performance includes, at least, for example: lower toxicity, stronger affinity for DNA and RNA without compromising sequence selectivity, improved pharmacokinetics and tissue distribution, improved cellular delivery, and both reliable and controllable in vivo distribution.

SUMMARY

Provided herein are peptide-oligonucleotide-conjugates comprising an oligonucleotide covalently bound to a bicyclic peptide. Also provided herein are methods of treating a disease in a subject in need thereof, comprising administering to the subject a peptide-oligonucleotide-conjugate described herein.

Accordingly, in one aspect, provided herein is a peptide-oligonucleotide-conjugate of Formula I:

or a pharmaceutically acceptable salt thereof,

wherein Formula I is covalently bound to a bicyclic peptide.

In one embodiment, the peptide-oligonucleotide-conjugate of Formula I is a peptide-oligonucleotide-conjugate of Formula Ia:

or a pharmaceutically acceptable salt thereof,

wherein (J)_(t)-G is a bicyclic peptide.

In another embodiment, the peptide-oligonucleotide-conjugate of Formula I is a peptide-oligonucleotide-conjugate of Formula Ib:

or a pharmaceutically acceptable salt thereof,

wherein (J)_(t)-G is a bicyclic peptide.

In still another aspect, provided herein is a method of treating a muscle disease, a viral infection, or a bacterial infection in a subject in need thereof, comprising administering to the subject a peptide-oligonucleotide-conjugate of the present disclosure.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows that cyclic and bicyclic peptides coupled to PMO-IVS2-654 (described in Example 6) demonstrate increased activity in a cellular exon-skipping assay. IVS2-654 corrects splicing of a mutated eGFP pre-mRNA. An increase in splice correction leads to an increase in fluorescence. Results have been normalized to the unconjugated IVS2-654 PMO.

FIG. 2 shows that bicyclic peptides demonstrate enhanced proteolytic stability relative to monocyclic peptides.

DETAILED DESCRIPTION

Provided herein are peptide-oligonucleotide-conjugates comprising an oligonucleotide covalently bound to a bicyclic peptide. Also provided herein are methods of treating a disease in a subject in need thereof; comprising administering to the subject a peptide-oligonucleotide-conjugate described herein. The oligonucleotides, and thereby the peptide-oligonucleotide-conjugates, described herein display stronger affinity for DNA and RNA without compromising sequence selectivity, relative to native or unmodified oligonucleotides. In some embodiments, the oligonucleotides of the disclosure minimize or prevent cleavage by RNase H. In some embodiments, the antisense oligonucleotides of the disclosure do not activate RNase H.

The peptides described herein impart to their corresponding peptide-oligonucleotide-conjugates lower toxicity, enhanced activity of the oligonucleotide, improve pharmacokinetics and tissue distribution, improve cellular delivery, and impart both reliable and controllable in vivo distribution.

DEFINITIONS

Listed below are definitions of various terms used to describe this disclosure. These definitions apply to the terms as they are used throughout this specification and claims, unless otherwise limited in specific instances, either individually or as part of a larger group.

The term “about” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. As used herein when referring to a measurable value such as an amount, a temporal duration, and the like, the term “about” is meant to encompass variations of ±20% or ±10%, including ±5%, ±1%, and ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

The term “alkyl” refers to saturated, straight- or branched-chain hydrocarbon moieties containing, in certain embodiments, between one and six, or one and eight carbon atoms, respectively. Examples of C₁₋₆-alkyl moieties include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, neopentyl, n-hexyl moieties; and examples of C₁₋₈-alkyl moieties include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, neopentyl, n-hexyl, heptyl, and octyl moieties.

The number of carbon atoms in an alkyl substituent can be indicated by the prefix “C_(x-y),” where x is the minimum and y is the maximum number of carbon atoms in the substituent. Likewise, a C_(x) chain means an alkyl chain containing x carbon atoms.

The term “heteroalkyl” by itself or in combination with another term means, unless otherwise stated, a stable straight or branched chain alkyl group consisting of the stated number of carbon atoms and one or two heteroatoms selected from the group consisting of O, N, and S, and wherein the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quatemized. The heteroatom(s) may be placed at any position of the heteroalkyl group, including between the rest of the heteroalkyl group and the fragment to which it is attached, as well as attached to the most distal carbon atom in the heteroalkyl group. Examples include: —O—CH₂—CH₂—CH₃, —CH₂—CH₂—CH₂—OH, —CH₂—CH₂—NH—CH₃, —CH₂—S—CH₂—Ch₃, and —CH₂—CH₂—S(═O)—CH₃. Up to two heteroatoms may be consecutive, such as, for example, —CH₂—NH—OCH₃, or —CH₂—CH₂—S—S—CH₃.

The term “aryl,” employed alone or in combination with other terms, means, unless otherwise stated, a carbocyclic aromatic system containing one or more rings (typically one, two, or three rings), wherein such rings may be attached together in a pendent manner, such as a biphenyl, or may be fused, such as naphthalene. Examples of aryl groups include phenyl, anthracyl, and naphthyl. In various embodiments, examples of an aryl group may include phenyl (e.g., C₆-aryl) and biphenyl (e.g., C₁₂-aryl). In some embodiments, aryl groups have from six to sixteen carbon atoms. In some embodiments, aryl groups have from six to twelve carbon atoms (e.g., C₆₋₁₂-aryl). In some embodiments, aryl groups have six carbon atoms (e.g., C₆-aryl).

As used herein, the term “heteroaryl” or “heteroaromatic” refers to a heterocycle having aromatic character. Heteroaryl substituents may be defined by the number of carbon atoms, e.g., C₁₋₉-heteroaryl indicates the number of carbon atoms contained in the heteroaryl group without including the number of heteroatoms. For example, a C₁₋₉-heteroaryl will include an additional one to four heteroatoms. A polycyclic heteroaryl may include one or more rings that are partially saturated. Non-limiting examples of heteroaryls include pyridyl, pyrazinyl, pyrimidinyl (including, e.g., 2- and 4-pyrimidinyl), pyridazinyl, thienyl, furyl, pyrrolyl (including, e.g., 2-pyrrolyl), imidazolyl, thiazolyl, oxazolyl, pyrazolyl (including, e.g., 3- and 5-pyrazolyl), isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl and 1,3,4-oxadiazolyl,

Non-limiting examples of polycyclic heterocycles and heteroaryls include indolyl (including, e.g., 3-, 4-, 5-, 6- and 7-indolyl), indolinyl, quinolyl, tetrahydroquinolyl, isoquinolyl (including, e.g., 1- and 5-isoquinolyl), 1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl (including, e.g., 2- and 5-quinoxalinyl), quinazolinyl, phthalazinyl, 1,8-naphthyridinyl, 1,4-benzodioxanyl, coumarin, dihydrocoumarin, 1,5-naphthyridinyl, benzofuryl (including, e.g., 3-, 4-, 5-, 6- and 7-benzofuryl), 2,3-dihydrobenzofuryl, 1,2-benzisoxazolyl, benzothienyl (including, e.g., 3-, 4-, 5-, 6-, and 7-benzothienyl), benzoxazolyl, benzothiazolyl(including, e.g., 2-benzothiazolyl and 5-benzothiazolyl), purinyl, benzimidazolyl (including, e.g., 2-benzimidazolyl), benzotriazolyl, thioxanthinyl, carbazolyl, carbolinyl, acridinyl, pyrrolizidinyl, and quinolizidinyl.

The term “protecting group” or “chemical protecting group” refers to chemical moieties that block some or all reactive moieties of a compound and prevent such moieties from participating in chemical reactions until the protective group is removed, for example, those moieties listed and described in T. W. Greene, P. G. M. Wuts, Protective Groups in Organic Synthesis, 3rd ed. John Wiley & Sons (1999). It may be advantageous, where different protecting groups are employed, that each (different) protective group be removable by a different means. Protective groups that are cleaved under totally disparate reaction conditions allow differential removal of such protecting groups. For example, protective groups can be removed by acid, base, and hydrogenolysis. Groups such as trityl, monomethoxytrityl, dimethoxytrityl, acetal and tert-butyldimethylsilyl are acid labile and may be used to protect carboxy and hydroxy reactive moieties in the presence of amino groups protected with Cbz groups, which are removable by hydrogenolysis, and Fmoc groups, which are base labile. Carboxylic acid moieties may be blocked with base labile groups such as, without limitation, methyl, or ethyl, and hydroxy reactive moieties may be blocked with base labile groups such as acetyl in the presence of amines blocked with acid labile groups such as tert-butyl carbamate or with carbamates that are both acid and base stable but hydrolytically removable.

Carboxylic acid and hydroxyl reactive moieties may also be blocked with hydrolytically removable protective groups such as the benzyl group, while amine groups may be blocked with base labile groups such as Fmoc. A particulary useful amine protecting group for the synthesis of compounds of Formula (I) is the trifluoroacetamide. Carboxylic acid reactive moieties may be blocked with oxidatively-removable protective groups such as 2,4-dimethoxybenzyl, while coexisting amino groups may be blocked with fluoride labile silyl carbamates.

Allyl blocking groups are useful in the presence of acid- and base-protecting groups since the former are stable and can be subsequently removed by metal or pi-acid catalysts. For example, an allyl-blocked carboxylic acid can be deprotected with a palladium(0)-catalyzed reaction in the presence of acid labile t-butyl carbamate or base-labile acetate amine protecting groups. Yet another form of protecting group is a resin to which a compound or intermediate may be attached. As long as the residue is attached to the resin, that functional group is blocked and cannot react. Once released from the resin, the functional group is available to react.

The term “nucleobase,” “base pairing moiety,” “nucleobase-pairing moiety,” or “base” refers to the heterocyclic ring portion of a nucleoside, nucleotide, and/or morpholino subunit. Nucleobases may be naturally occurring, or may be modified or analogs of these naturally occurring nucleobases, e.g., one or more nitrogen atoms of the nucleobase may be independently at each occurrence replaced by carbon. Exemplary analogs include hypoxanthine (the base component of the nucleoside inosine); 2,6-diaminopurine; 5-methyl cytosine; C5-propynyl-modified pyrimidines; 10-(9-(aminoethoxy)phenexazinyl) (G-clamp) and the like.

Further examples of base pairing moieties include, but are not limited to, uracil, thymine, adenine, cytosine, guanine and hypoxanthine having their respective amino groups protected by acyl protecting groups, 2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5-iodouracil, 2,6-diaminopurine, azacytosine, pyrimidine analogs such as pseudoisocytosine and pseudouracil and other modified nucleobases such as 8-substituted purines, xanthine, or hypoxanthine (the latter two being the natural degradation products). The modified nucleobases disclosed in Chiu and Rana, RNA, 2003, 9, 1034-1048, Limbach et al. Nucleic Acids Research, 1994, 22, 2183-2196 and Revankar and Rao, Comprehensive Natural Products Chemistry, vol. 7, 313, are also contemplated, the contents of which are incorporated herein by reference.

Further examples of base pairing moieties include, but are not limited to, expanded-size nucleobases in which one or more benzene rings has been added. Nucleic base replacements described in the Glen Research catalog (www.glenresearch.com); Krueger A T et al., Acc. Chem. Res., 2007, 40, 141150; Kool, E T, Acc. Chem. Res., 2002, 35, 936-943; Benner S. A., et al., Nat. Rev. Genet., 2005, 6, 553-543; Romesberg, F. E., et al., Curr. Opin. Chem. Biol., 2003, 7, 723-733; Hirao, I., Curr. Opin. Chem. Biol., 2006, 10, 622-627, the contents of which are incorporated herein by reference, are contemplated as useful for the synthesis of the oligomers described herein. Examples of expanded-size nucleobases are shown below:

The terms “oligonucleotide” or “oligomer” refer to a compound comprising a plurality of linked nucleosides, nucleotides, or a combination of both nucleosides and nucleotides. In specific embodiments provided herein, an oligonucleotide is a morpholino oligonucleotide.

The phrase “morpholino oligonucleotide” or “PMO” refers to a modified oligonucleotide having morpholino subunits linked together by phosphoramidate or phosphorodiamidate linkages, joining the morpholino nitrogen of one subunit to the 5′-exocyclic carbon of an adjacent subunit. Each morpholino subunit comprises a nucleobase-pairing moiety effective to bind, by nucleobase-specific hydrogen bonding, to a nucleobase in a target.

The terms “antisense oligomer,” “antisense compound” and “antisense oligonucleotide” are used interchangeably and refer to a sequence of subunits, each bearing a base-pairing moiety, linked by intersubunit linkages that allow the base-pairing moieties to hybridize to a target sequence in a nucleic acid (typically an RNA) by Watson-Crick base pairing, to form a nucleic acid:oligomer heteroduplex within the target sequence. The oligomer may have exact (perfect) or near (sufficient) sequence complementarity to the target sequence; variations in sequence near the termini of an oligomer are generally preferable to variations in the interior.

Such an antisense oligomer can be designed to block or inhibit translation of mRNA or to inhibit/ether natural or abnormal pre-mRNA splice processing, and may be said to be “directed to” or “targeted against” a target sequence with which it hybridizes. The target sequence is typically a region including an AUG start codon of an mRNA, a Translation Suppressing Oligomer, or splice site of a pre-processed mRNA, a Splice Suppressing Oligomer (SSO). The target sequence for a splice site may include an mRNA sequence having its 5′ end 1 to about 25 base pairs downstream of a normal splice acceptor junction in a preprocessed mRNA. In various embodiments, a target sequence may be any region of a preprocessed mRNA that includes a splice site or is contained entirely within an exon coding sequence or spans a splice acceptor or donor site. An oligomer is more generally said to be “targeted against” a biologically relevant target, such as a protein, virus, or bacteria, when it is targeted against the nucleic acid of the target in the manner described above.

The antisense oligonucleotide and the target RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other, such that stable and specific binding occurs between the oligonucleotide and the target. Thus, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the target. It is understood in the art that the sequence of an oligonucleotide need not be 100% complementary to that of its target sequence to be specifically hybridizable. An oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target molecule interferes with the normal function of the target RNA, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense oligonucleotide to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed.

Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. Oligonucleotides containing a modified or substituted base include oligonucleotides in which one or more purine or pyrimidine bases most commonly found in nucleic acids are replaced with less common or non-natural bases. In some embodiments, the nucleobase is covalently linked at the N9 atom of the purine base, or at the N1 atom of the pyrimidine base, to the morpholine ring of a nucleotide or nucleoside.

Purine bases comprise a pyrimidine ring fused to an imidazole ring, as described by the general formula:

Adenine and guanine are the two purine nucleobases most commonly found in nucleic acids. These may be substituted with other naturally-occurring purines, including but not limited to N6-methyladenine, N2-methylguanine, hypoxanthine, and 7-methylguanine.

Pyrimidine bases comprise a six-membered pyrimidine ring as described by the general formula:

Cytosine, uracil, and thymine are the pyrimidine bases most commonly found in nucleic acids. These may be substituted with other naturally-occurring pyrimidines, including but not limited to 5-methylcytosine, 5-hydroxymethylcytosine, pseudouracil, and 4-thiouracil. In one embodiment, the oligonucleotides described herein contain thymine bases in place of uracil.

Other modified or substituted bases include, but are not limited to, 2,6-diaminopurine, orotic acid, agmatidine, lysidine, 2-thiopyrimidine (e.g. 2-thiouracil, 2-thiothymine), G-clamp and its derivatives, 5-substituted pyrimidine (e.g. 5-halouracil, 5-propynyluracil, 5-propynyleytosine, 5-aminomethyluracil, 5-hydroxymethyluracil, 5-aminomethylcytosine, 5-hydroxymethylcytosine, Super T), 7-deazaguanine, 7-deazaadenine, 7-aza-2,6-diaminopurine, 8-aza-7-deazaguanine, 8-aza-7-deazaadenine, 8-aza-7-deaza-2,6-diaminopurine, Super G, Super A, and N4-ethylcytosine, or derivatives thereof; N2-cyclopentylguanine (cPent-G), N2-cyclopentyl-2-aminopurine (ePent-AP), and N2-propyl-2-aminopurine (Pr-AP), pseudouracil or derivatives thereof; and degenerate or universal bases, like 2,6-difiuorotoluene or absent bases like abasic sites (e.g. 1-deoxyribose, 1,2-dideoxyribose, 1-deoxy-2-O-methylribose; or pyrrolidine derivatives in which the ring oxygen has been replaced with nitrogen (azaribose)). Pseudouracil is a naturally occuring isomerized version of uracil, with a C-glycoside rather than the regular N-glycoside as in uridine.

Certain modified or substituted nucleobases are particularly useful for increasing the binding affinity of the antisense oligonucleotides of the disclosure. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. In various embodiments, nucleobases may include 5-methylcytosine substitutions, which have been shown to increase nucleic acid duplex stability by 0.6-1.2° C.

In some embodiments, modified or substituted nucleobases are useful for facilitating purification of antisense oligonucleotides. For example, in certain embodiments, antisense oligonucleotides may contain three or more (e.g. , 3, 4, 5, 6 or more) consecutive guanine bases. In certain antisense oligonucleotides, a string of three or more consecutive guanine bases can result in aggregation of the oligonucleotides, complicating purification. In such antisense oligonucleotides, one or more of the consecutive guanines can be substituted with hypoxanthine. The substitution of hypoxanthine for one or more guanines in a string of three or more consecutive guanine bases can reduce aggregation of the antisense oligonucleotide, thereby facilitating purification.

The oligonucleotides provided herein are synthesised and do not include antisense compositions of biological origin. The molecules of the disclosure may also be mixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution, or absorption, or a combination thereof.

The terms “complementary” and “complementarity” refer to oligonucleotides (i.e., a sequence of nucleotides) related by base-pairing rules. For example, the sequence “T-G-A (5′-3),” is complementary to the sequence “T-C-A (5′-3′).” Complementarity may be “partial,” in which only some of the nucleic acids' bases are matched according to base pairing rules. Or, there may be “complete,” “total,” or “perfect” (100%) complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. While perfect complementarity is often desired, some embodiments can include one or more but preferably 6, 5, 4, 3, 2, or 1 mismatches with respect to the target RNA. Such hybridization may occur with “near” or “substantial” complementarity of the antisense oligomer to the target sequence, as well as with exact complementarity. In some embodiments, an oligomer may hybridize to a target sequence at about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% complementarity. Variations at any location within the oligomer are included. In certain embodiments, variations in sequence near the termini of an oligomer are generally preferable to variations in the interior, and if present are typically within about 6, 5, 4, 3, 2, or 1 nucleotides of the 5′-terminus, 3′-terminus, or both termini.

The term “peptide” refers to a compound comprising a plurality of linked amino acids. The peptides provided herein can be considered to be cell penetrating peptides.

The terms “cell penetrating peptide” and “CPP” are used interchangeably and refer to cationic cell penetrating peptides, also called transport peptides, carrier peptides, or peptide transduction domains. The peptides, provided herein, have the capability of inducing cell penetration within 100% of cells of a given cell culture population and allow macromolecular translocation within multiple tissues in vivo upon systemic administration. In various embodiments, a CPP embodiment of the disclosure may include an arginine-rich peptide as described further below.

The term “treatment” refers to the application of one or more specific procedures used for the amelioration of a disease. In certain embodiments, the specific procedure is the administration of one or more pharmaceutical agents. “Treatment” of an individual (e.g. a mammal, such as a human) or a cell is any type of intervention used in an attempt to alter the natural course of the individual or cell. Treatment includes, but is not limited to, administration of a pharmaceutical composition, and may be performed either prophylactically or subsequent to the initiation of a pathologic event or contact with an etiologic agent. Treatment includes any desirable effect on the symptoms or pathology of a disease or condition, and may include, for example, minimal changes or improvements in one or more measurable markers of the disease or condition being treated. Also included are “prophylactic” treatments, which can be directed to reducing the rate of progression of the disease or condition being treated, delaying the onset of that disease or condition, or reducing the severity of its onset.

An “effective amount” or “therapeutically effective amount” refers to an amount of therapeutic compound, such as an antisense oligomer, administered to a mammalian subject, either as a single dose or as part of a series of doses, which is effective to produce a desired therapeutic effect.

The term “amelioration” means a lessening of severity of at least one indicator of a condition or disease. In certain embodiments, amelioration includes a delay or slowing in the progression of one or more indicators of a condition or disease. The severity of indicators may be determined by subjective or objective measures which are known to those skilled in the art.

As used herein, “pharmaceutically acceptable salts” refers to derivatives of the disclosed oligonucleotides wherein the parent oligonucleotide is modified by converting an existing acid or base moiety to its salt form, Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977), each of which is incorporated herein by reference in its entirety.

Peptide-Oligonucleotide-Conjugates

Provided herein are oligonucleotides chemically linked to a bicyclic, cell-penetrating peptide. The bicyclic, cell-penetrating peptide enhances activity, cellular distribution, or cellular uptake of the oligonucleotide. In some embodiments, the CPP can be an arginine-rich peptide. The oligonucleotides can additionally be chemically-linked to one or more heteroalkyl moieties (e.g., polyethylene glycol) that further enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. In one exemplary embodiment, the polypeptide, e.g., the arginine-rich polypeptide, is covalently coupled at its N-terminal or C-terminal residue to either end, or both ends, of the oligonucleotide.

Thus, in one aspect, provided herein is a peptide-oligonucleotide-conjugate of Formula I:

or a pharmaceutically acceptable salt thereof,

wherein:

A′ is selected from —NHCH₂C(O)NH₂, —N(C₁₋₆-alkyl)CH₂C(O)NH₂,

wherein

R⁵ is —C(O)(O-alkyl)_(x)—OH, wherein x is 3-10 and each alkyl group is independently at each occurrence C₂₋₆-alkyl, or R⁵ is selected from —C(O)C₁₋₆-alkyl, trityl, monomethoxytrityl, -(C₁₋₆-alkyl)R⁶, -(C₁₋₆-heteroalkyl)-R⁶, aryl-R⁶, heteroaryl-R⁶, —C(O)O-(C₁₋₆-alkyl)-R⁶, —C(O)O-aryl-R⁶, —C(O)O-heteroaryl-R⁶, and

wherein R⁶ is selected from OH, SH, and NH₂, or R⁶ is O, S, or NH, covalently linked. to a solid support;

each R¹ is independently selected from OH and —NR³R⁴, wherein each R³ and R⁴ are independently at each occurrence -C₁₋₆-alkyl;

each R² is independently selected from H, a nucleobase, and a nucleobase functionalized with a chemical protecting-group, wherein the nucleobase independently at each occurrence comprises a C₃₋₆-heterocyclic ring selected from pyridine, pyrimidine, triazinane, purine, and deaza-purine;

z is 8-40; and

E′ is selected from H, -C₁₋₆-alkyl, benzoyl, stearoyl, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl,

wherein

Q is —C(O)(CH₂)₆C(O)— —C(O)(CH₂)₂S₂(CH₂)₂C(O)—, and

R7 is —(CH₂)₂OC(O)N(R⁸)₂, wherein R⁸ is —(CH₂)₆NHC(═NH)NH₂, and

wherein L is covalently linked by an amide bond to the amino-terminus of J, and L is —C(O)(CH₂)₁₋₆-C₁₋₆-heteroaromatic-(CH₂)₁₋₆C(O);

t is 4-16;

each J is independently at each occurrence selected from an amino acid of the structure

wherein:

r and q are each independently 0, 1, 2, 3, or 4; and

each R⁹ is independently at each occurrence selected from H, an amino acid side-chain, and an amino acid side-chain functionalized with a protecting-group,

wherein three or four amino acid side-chains of R⁹ independently at each occurrence comprise a sulfur atom:

a) wherein when the number of sulfur atoms is three, then the sulfur atoms, together with the atoms to which they are attached, form the structure

such that the amino acids form a bicyclic structure; or

b) wherein when the number of sulfur atoms is four, then the sulfur atoms, together with the atoms to which they are attached, form two occurrences of the structure

such that the amino acids form a bicyclic structure;

wherein d is 0 or 1, and M is selected from:

wherein each R¹ is independently at each occurrence H or a halogen; and

G is covalently linked by an amide bond to the carboxy-terminus of J, and G is selected from H, —C(O)C₁₋₆-alkyl, benzoyl, and stearoyl, and

wherein at least one of the following conditions is true:

-   -   1) A′ is

or 2) E′ is

In one embodiment, E′ is selected from H, -C₁₋₆-alkyl, —C(O)C₁₋₆-alkyl, benzoyl, stearoyl, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, and

In yet another embodiment, A′ is selected from —N(C₁₋₆-alkyl)CH₂C(O)NH₂,

In another embodiment E′ is selected from H, —C(O)CH₃, benzoyl, stearoyl, trityl, 4-methoxytrityl, and

In still another embodiment, A′ is selected from —N(C₁₋₆-alkyl)CH₂C(O)NH₂,

and

-   -   E′ is

In yet another embodiment, A′ is

In another embodiment, E′ is selected from H, —C(O)CH₃, trityl, 4-methoxytrityl, benzoyl, and stearoyl.

In yet another embodiment, E′ is selected from H and —C(O)CH₃.

In still another embodiment, the peptide-oligonucleotide-conjugate of Formula I is a peptide-oligonucleotide-conjugate of Formula Ia:

In another embodiment, the peptide-oligonucleotide-conjugate of Formula I is a peptide-oligonucleotide-conjugate of Formula Ib:

In an embodiment of Formula I and Ib, E′ is selected from H, C₁₋₆ alkyl, —C(O)CH₃, benzoyl, and stearoyl.

In another embodiment of Formula I and Ib, E′ is selected from H and —C(O)CH₃.

In an embodiment of Formula I, Ia, and Ib, each R¹⁰ is independently a halogen selected from fluorine, chlorine, bromine, and iodine.

In another embodiment of Formula I, Ia, and Ib, each R¹⁰ is fluorine.

In still another embodiment of Formula I, Ia, and Ib, M is

In another embodiment of Formula I, Ia, and Ib, three amino acid side-chain groups are independently at each occurrence cysteine or homocysteine amino acid side-chain groups.

In another embodiment of Formula I, Ia, and Ib, four amino acid side-chain groups are independently at each occurrence cysteine or homocysteine amino acid side-chain groups.

In still another embodiment of Formula. I, Ia, and Ib, each J is independently at each occurrence selected from an α-amino acid, a β²-amino acid, and a β³-amino acid.

In yet another embodiment of Formula I, Ia, and Ib, r and q are each 0.

In another embodiment of Formula I, Ia, and Ib, J is independently selected from cysteine and arginine.

In still another embodiment of Formula I, Ia, and Ib, three J groups are cysteine.

In still another embodiment of Formula I, Ia, and Ib, four J groups are cysteine.

In yet another embodiment of Formula I, Ia, and Ib, z is 9-16.

In another embodiment of Formula I, Ia, and Ib, each R¹ is independently NR³R⁴, wherein each R³ and R⁴ are independently at each occurrence C₁₋₃-alkyl.

In still another embodiment of Formula I, Ia, and Ib, each R¹ is N(CH₃)₂.

In yet another embodiment of Formula I, Ia, and Ib, each R² is a nucleobase, wherein the nucleobase independently at each occurrence comprises a C₄₋₆-heterocyclic ring selected from pyridine, pyrimidine, triazinane, purine, and deaza-purine.

In another embodiment of Formula I, Ia, and Ib, each R² is a nucleobase, wherein the nucleobase independently at each occurrence comprises a C₄₋₆-heterocyclic ring selected from pyrimidine, purine, and deaza-purine.

In still another embodiment of Formula I, Ia, and Ib, each R² is a nucleobase independently at each occurrence selected from adenine, 2,6-diaminopurine, 7-deaza-adenine, guanine, 7-deaza-guanine, hypoxanthine, cytosine, 5-methyl-cytosine, thymine, uracil, and hypoxanthine.

In yet another embodiment of Formula I, Ia, and Ib, each R² is a nucleobase independently at each occurrence selected from adenine, guanine, cytosine, 5-methyl-cytosine, thymine, uracil, and hypoxanthine.

In another embodiment of Formula I, Ia, and Ib, L is

In another embodiment of Formula Ia, and Ib, G is selected from H, C(O)CH₃, benzoyl, and stearoyl.

In still another embodiment of Formula I, Ia, and Ib, G is H or —C(O)CH₃.

In yet another embodiment of Formula I, Ia, and Ib, G is H.

In yet another embodiment of Formula I, Ia, and Ib, G is —C(O)CH₃.

In another embodiment of Formula I, Ia, and Ib, d is 1.

In still another embodiment of Formula I, Ia, and Ib, d is 0.

In yet another embodiment of Formula I, Ia, and Ib,

is selected from:

wherein [L] is

X is, at each occurrence, independently selected from arginine or cysteine;

C is cysteine;

n is an integer selected from 3-8; and

m is an integer selected from 3-8, or

wherein [L] is

X is, at each occurrence, independently selected from arginine or cysteine;

C is cysteine;

G is selected from H, C(O)CH₃, benzoyl, and stearoyl, wherein G is covalently linked by an amide bond to the carboxy terminus of the peptide;

n is an integer selected from 3-8; and

m is an integer selected from 3-8.

In yet another embodiment of Formula I, Ia, and Ib,

is selected from:

wherein X is, at each occurrence, independently selected from arginine or cysteine;

C is cysteine; and

G is selected from H, C(O)CH₃, benzoyl, and stearoyl, wherein G is covalently linked by an amide bond to the carboxy terminus of the peptide.

In an embodiment, at least one instance of X is arginine.

In another embodiment, at least two instances of X are arginine.

In yet another embodiment, at least three instances of X are arginine.

In an embodiment, at least four instances of X are arginine.

In a further embodiment, at least five instances of X are arginine.

In yet another embodiment, at least six instances of X are arginine.

In an embodiment, at least seven instances of X are arginine.

In yet another embodiment of Formula I, Ia, and Ib,

is selected from:

wherein R is arginine and C is cysteine; and

G is selected from H, C(O)CH₃, benzoyl, and stearoyl, wherein G is covalently linked by an amide bond to the carboxy terminus of the peptide.

In yet another embodiment, the oligonucleotide comprises a targeting sequence having sequence complementarity to an RNA target. In a specific embodiment, the RNA target is a cellular RNA target. In another specific embodiment, the targeting sequence has sufficient sequence complementarity to bind to the RNA target. In yet another specific embodiment, the targeting sequence has perfect sequence complementarity to the RNA target.

Representative peptide-oligonucleotide-conjugates of the disclosure include, amongst others, peptide-oligonucleotide-conjugates of the following structures:

or a pharmaceutically acceptable salt thereof, wherein

G is H or —C(O)CH₃, wherein G is covalently linked by an amide bond to the carboxy terminus of the peptide;

C is cysteine;

R is arginine;

R² is a nucleobase, independently at each occurrence, selected from adenine, guanine, cytosine, 5-methyl-cytosine, thymine, uracil, and hypoxanthine; and

z is 8-40.

In one embodiment of the peptide-oligonucleotide-conjugates of the disclosure, G is H.

In another embodiment of the peptide-oligonucleotide-conjugates of the disclosure, G is —C(O)CH₃.

In an embodiment, L is covalently linked to the carboxy terminus of the peptide, and G is covalently linked to the amino terminus of the peptide.

As used herein, “G is covalently linked by an amide bond to the carboxy-terminus of J” indicates that the carboxy-terminus of J (—COOH) is covalently bound to variable G via an N(H) group, wherein the hydroxyl group of the carboxy-terminus of J is replaced with N(H). For example, when G is H, the following structure is formed by J and G:

In some embodiments, the peptide-oligonucleotide-conjugates described herein are unsolvated. In other embodiments, one or more of the peptide-oligonucleotide-conjugates are in solvated form. As known in the art, the solvate can be any of pharmaceutically acceptable solvent, such as water, ethanol, and the like.

Although the peptide-oligonucleotide-conjugates of Formulae I, Ia, Ib, Ic, Id, Ie, and IV are depicted in their neutral forms, in some embodiments, these peptide-oligonucleotide-conjugates are used in a pharmaceutically acceptable salt form.

Oligonucleotides

Important properties of morpholino-based subunits include: 1) the ability to be linked in a oligomeric form by stable, uncharged or positively charged backbone linkages; 2) the ability to support a nucleotide base (e.g. adenine, cytosine, guanine, thymidine, uracil, 5-methyl-cytosine and hypoxanthine) such that the polymer formed can hybridize with a complementary-base target nucleic acid, including target RNA, T_(M) values above about 45° C. in relatively short oligonucleotides (e.g. , 10-15 bases); 3) the ability of the oligonucleotide to be actively or passively transported into mammalian cells; and 4) the ability of the oligonucleotide and oligonucleotide:RNA heteroduplex to resist RNAse and RNase H degradation, respectively.

The stability of the duplex formed between an oligomer and a target sequence is a function of the binding T_(M) and the susceptibility of the duplex to cellular enzymatic cleavage. The T_(M) of an oligomer with respect to complementary-sequence RNA may be measured by conventional methods, such as those described by Hames et al., Nucleic Acid Hybridization, IRL Press, 1985, pp. 107-108 or as described in Miyada C. G. and Wallace R. B., 1987, Oligomer Hybridization Techniques, Methods Enzymol. Vol. 154 pp. 94-107. In certain embodiments, antisense oligomers may have a binding T_(M), with respect to a complementary-sequence RNA, of greater than body temperature and, in some embodiments greater than about 45° C. or 50° C. T_(M)'s in the range 60-80° C. or greater are also included. According to well-known principles, the T_(M) of an oligomer, with respect to a complementary-based RNA hybrid, can be increased by increasing the ratio of C:G paired bases in the duplex, or by increasing the length (in base pairs) of the heteroduplex, or both. At the same time, for purposes of optimizing cellular uptake, it may be advantageous to limit the size of the oligomer. For this reason, compounds of the disclosure include compounds that show a high T_(M) (45-50° C. or greater) at a length of 25 bases or less.

The length of an oligonucleotide may vary so long as it is capable of binding selectively to the intended location within the pre-mRNA molecule. The length of such sequences can he determined in accordance with selection procedures described herein. Generally, the oligonucleotide will be from about 8 nucleotides in length up to about 50 nucleotides in length. For example, the length of the oligonucleotide (z) can be 8-38, 8-25, 15-25, 17-21, or about 18. It will be appreciated however that any length of nucleotides within this range may be used in the methods described herein.

In some embodiments, the antisense oligonucleotides contain base modifications or substitutions. For example, certain nucleo-bases may be selected to increase the binding affinity of the antisense oligonucleotides described herein. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aininopropyladenine, 5-propynyluracil, 5-propynylcytosine and 2,6-diaminopurine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C., and may be incorporated into the antisense oligonucleotides described herein. In one embodiment, at least one pyrimidine base of the oligonucleotide comprises a 5-substituted pyrimidine base, wherein the pyrimidine base is selected from the group consisting of cytosine, thymine and uracil. In one embodiment, the 5-substituted pyrimidine base is 5-methylcytosine. In another embodiment, at least one purine base of the oligonucleotide comprises an N-2, N-6 substituted purine base. In one embodiment, the N-2, N-6 substituted purine base is 2, 6-diaminopurine.

Morpholino-based oligomers (including antisense oligomers) are detailed, for example, in U.S. Pat. Nos. 5,698,685; 5,217,866; 5,142,047; 5,034,506; 5,166,315; 5,185,444; 5,521,063; 5,506,337 and pending U.S. patent application Ser. Nos. 12/271,036; 12/271,040; and PCT Publication No. WO/2009/064471 and WO/2012/043730 and Summerton et al. 1997, Antisense and Nucleic Acid Drug Development, 7, 187-195, which are hereby incorporated by reference in their entirety.

Accordingly, in one aspect, provided herein is an oligonucleotide of Formula II:

or a pharmaceutically acceptable salt thereof,

wherein

A is selected from the group consisting of OH, —NHCH₂C(O)NH₂, —N(C₁₋₆-alkyl)CH₂C(O)NH₂,

R⁵ is —C(O)(O-alkyl)_(x)OH, wherein x is 3-10 and each alkyl group is independently at each occurrence -C₂₋₆-alkyl, or R⁵ is selected from the group consisting of —C(O)C₁₋₆-alkyl, trityl, monomethoxytrityl, -C₁₋₆-alkyl-R⁶, -C₁₋₆heteroalkyl-R⁶, -aryl-R⁶, -heteroaryl-R⁶, —C(O)O-C₁₋₆-alkyl-R⁶, —C(O)O-aryl-R⁶, and —C(O)O-heteroaryl-R⁶;

R⁶ is selected from the group consisting of OH, SH, and NH₂, or R⁶ is O, S, or NH, covalently linked to a solid support;

each R¹ is independently OH or —NR³R⁴;

each R³ and R⁴ are independently at each occurrence -C₁₋₆-alkyl;

each R² is independently selected from the group consisting of H, a nucleobase, and a nucleobase functionalized with a chemical protecting-group, wherein the nucleobase independently at each occurrence comprises a C₃₋₆-heterocyclic ring selected from the group consisting of pyridine, pyrimidine, triazinane, purine, and deaza-purine;

z is 8-40;

E is selected from the group consisting of H, -C₁₋₆-alkyl, —C(O)C₁₋₆-alkyl, benzoyl, stearoyl, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, and

Q is —C(O)(CH₂)₆C(O)— or —C(O)(CH₂)₂S₂(CH₂)₂C(O)—;

R⁷ is —(CH₂)₂OC(O)N(R⁸)₂;

R⁸ is —(CH₂)₆NHC(═NH)NH₂.

In one embodiment of Formula II, A is

E is selected from the group consisting of H, —C(O)CH₃, benzoyl, and stearoyl;

R⁵ is —C(O)(O-alkyl)_(x)-OH, wherein each alkyl group is independently at each occurrence -C₂₋₆-alkyl, trityl, and 4-methoxytrityl; and

each R² is independently a nucleobase, wherein the nucleobase independently at each occurrence comprises a C₄₋₆-heterocyclic ring selected from the group consisting of pyridine, pyrimidine, purine, and deaza-purine.

In another embodiment of Formula II, R⁵ is C(O)(O—CH₂CH₂)₃—OH; and

each R² is independently a nucleobase, wherein the nucleobase independently at each occurrence comprises a pyrimidine or a purine.

In still another embodiment, the oligonucleotide of Formula II is an oligonucleotide of Formula IIa:

In an embodiment of Formula II and IIa, R² is independently at each occurrence adenine, 2,6-diaminopurine, guanine, hypoxanthine, cytosine, 5-methyl-cytosine, thymine, uracil, and hypoxanthine; and

each R¹ is —N(CH₃)₂.

Provided in Table 1 are various embodiments of nucleotide moieties as described herein.

TABLE 1 Various embodiments of nucleotide moieties.

In some embodiments, the oligonucleotides described herein are unsolvated. In other embodiments, one or more of the oligonucleotides are in solvated form. As known in the art, the solvate can be any of pharmaceutically acceptable solvent, such as water, ethanol, and the like.

Although the oligonucleotides of Formulas II and IIa, are depicted in their neutral forms, in some embodiments, these oligonucleotides are used in a pharmaceutically acceptable salt form.

Peptides

The oligonucleotides provided herein include an oligonucleotide moiety conjugated to a CPP, wherein the CPP comprises a bicyclic peptide. In some embodiments, the CPP can be an arginine-rich peptide transport moiety effective to enhance transport of the compound into cells. The transport moiety is, in some embodiments, attached to a terminus of the oligomer. The peptides have the capability of inducing cell penetration within 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of cells of a given cell culture population, including all integers in between, and allow macromolecular translocation within multiple tissues in vivo upon systemic administration. In one embodiment, the cell-penetrating peptide may be an arginine-rich peptide transporter. In various embodiments, a peptide-oligonucleotide-conjugate of the present disclosure may utilize glycine as the linker between the CPP and the antisense oligonucleotide.

The transport moieties as described above have been shown to greatly enhance cell entry of attached oligomers, relative to uptake of the oligomer in the absence of the attached transport moiety. Uptake may be enhanced at least ten fold, and, in some embodiments, twenty fold, relative to the unconjugated compound.

The use of arginine-rich peptide transporters (i.e., cell-penetrating peptides) are particularly useful in practicing the present disclosure. Certain peptide transporters have been shown to be highly effective at delivery of antisense compounds into primary cells including muscle cells. Furthermore, compared to other known peptide transporters such as Penetratin and the Tat peptide, the peptide transporters described herein, when conjugated to an antisense PMO, demonstrate an enhanced ability to alter splicing of several gene transcripts.

Thus, in one aspect, provided herein is a peptide of Formula III:

G-(J)_(t)-L  (III)

or a pharmaceutically acceptable salt thereof,

wherein

G is selected from the group consisting of H, C(O)C₁₋₆-alkyl, benzoyl, and stearoyl, wherein G is covalently linked by an amide bond to the carboxy terminus of the peptide;

each J is independently at each occurrence selected from an amino acid of the structure

each R⁹ is independently at each occurrence selected from the group consisting of H, an amino acid side-chain, and an amino acid side-chain functionalized with a chemical protecting-group;

wherein three or four amino acid side-chain groups of R⁹ independently at each occurrence comprise a thiol or a thiol functionalized with a chemical protecting-group;

r and q are independently 0, 1, 2, 3, or 4;

L is —C(O)(CH₂)₁₋₆-C₁₋₆-heteroaromatic-(CH₂)₁₋₆C(O), which may be covalently-linked to a solid support; and

t is 4-16.

In one embodiment, three or four amino acid side-chains of R⁹ independently at each occurrence comprise a sulfur atom:

a) wherein when the number of sulfur atoms is three, then the sulfur atoms, together with the atoms to which they are attached, form the structure

such that the amino acids form a bicyclic structure; or b) wherein when the number of sulfur atoms is four, then the sulfur atoms, together with the atoms to which they are attached, form two occurrences of the structure

such that the amino acids form a bicyclic structure;

-   -   wherein d is 0 or 1, and M is selected from:

-   -   wherein each R¹⁰ is independently at each occurrence H or a         halogen.

In another embodiment, M is

In yet another embodiment, three amino acid side-chain groups are independently at each occurrence cysteine or homocysteine amino acid side-chain groups.

In yet another embodiment, four amino acid side-chain groups are independently at each occurrence cysteine or homocysteine amino acid side-chain groups.

In still another embodiment, each J is independently at each occurrence selected from an α-amino acid, a β²-amino acid, and a β³-amino acid.

In another embodiment, r and q are each 0.

In another embodiment, J is independently selected from cysteine and arginine.

In yet another embodiment, three J groups are cysteine.

In yet another embodiment, four J groups are cysteine.

In still another embodiment, L is —C(O)(CH₂)₁₋₆-C₁₋₆-heteroaromatic-(CH₂)₁₋₆C(O)

In another embodiment, L is

In yet another embodiment, G is selected from the group consisting of H, C(O)CH₃, benzoyl, and stearoyl.

In still another embodiment, G is C(O)CH₃ or H.

In another embodiment, G is C(O)CH₃.

In another embodiment, G is H.

In yet another embodiment, G is covalently linked by an amide bond to the carboxy-terminus of J. In a further embodiment, L is covalently linked by an amide bond to the amino-terminus of J.

In another embodiment, d is 0.

In yet another embodiment, d is 1.

In some embodiments, the peptides described herein are unsolvated. In other embodiments, one or more of the peptides are in solvated form. As known in the art, the solvate can be any of pharmaceutically acceptable solvent, such as water, ethanol, and the like.

Although the peptides of Formula III, are depicted in their neutral forms, in some embodiments, these oligonucleotides are used in a pharmaceutically acceptable salt form.

Methods

Provided herein are methods of treating a muscle disease, a viral infection, or a bacterial infection in a subject in need thereof, comprising administering to the subject a peptide-oligonucleotide-conjugate of Formulae I, Ia, or Ib.

Accordingly, in one aspect, provided herein is a method of treating a muscle disease, a viral infection, or a bacterial infection in a subject in need thereof; comprising administering to the subject a peptide-oligonucleotide-conjugate of the present disclosure.

In one embodiment, the muscle disease is Duchenne Muscular Dystrophy.

In another embodiment, the viral infection is caused by a virus selected from the group consisting of marburg virus, ebola virus, influenza virus, and dengue virus.

In another embodiment, the bacterial infection is caused by Mycobacterium tuberculosis.

The subject considered herein is typically a human. However, the subject can be any mammal for which treatment is desired. Thus, the methods described herein can be applied to both human and veterinary applications.

Administration/Dose

The formulation of therapeutic compositions and their subsequent administration (dosing) is within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a sufficient diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient.

Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligomers, and can generally be estimated based on EC_(50S) found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 μg to 100 g/kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligomer is administered in maintenance doses, ranging from 0.01 μg to 100 g/kg of body weight, once or more daily, to once every 20 years.

In some embodiments, the oligonucleotide (an oligonucleotide of Formulae II or IIa) is administered alone.

In some embodiments, the oligonucleotide is administered in a therapeutically effective amount or dosage. A “therapeutically effective amount” is an amount of an oligonucleotide of Formula II or IIa that, when administered to a patient by itself; effectively treats a muscle disease, a viral infection, or a bacterial infection. An amount that proves to be a “therapeutically effective amount” in a given instance, for a particular subject, may not be effective for 100% of subjects similarly treated for the disease or condition under consideration, even though such dosage is deemed a “therapeutically effective amount” by skilled practitioners. The amount of the oligonucleotide that corresponds to a therapeutically effective amount is strongly dependent on the type of disease, stage of the disease, the age of the patient being treated, and other facts.

In different embodiments, depending on the oligonucleotide of Formulae II or IIa and the effective amounts used, the oligonucleotides can modulate the expression of a gene involved in a muscle disease, a viral infection, or a bacterial infection.

While the amounts of an oligonucleotide of Formulae II or IIa should result in the effective treatment of a muscle disease, a viral infection, or a bacterial infection, the amounts, are preferably not excessively toxic to the patient (i.e., the amounts are preferably within toxicity limits as established by medical guidelines). In some embodiments, either to prevent excessive toxicity or provide a more efficacious treatment, or both, of a muscle disease, a viral infection, or a bacterial infection, a limitation on the total administered dosage is provided. Typically, the amounts considered herein are per day; however, half-day and two-day or three-day cycles also are considered herein.

Different dosage regimens may be used to treat a muscle disease, a viral infection, or a bacterial infection. In some embodiments, a daily dosage, such as any of the exemplary dosages described above, is administered once, twice, three times, or four times a day for three, four, five, six, seven, eight, nine, or ten days. Depending on the stage and severity of the disease being treated, a shorter treatment time (e.g., up to five days) may be employed along with a high dosage, or a longer treatment time (e.g., ten or more days, or weeks, or a month, or longer) may be employed along with a low dosage. In some embodiments, a once-or twice-daily dosage is administered every other day.

Oligonucleotides of Formula II and IIa, or their pharmaceutically acceptable salts or solvate forms, in pure form or in an appropriate pharmaceutical composition, can be administered via any of the accepted modes of administration or agents known in the art. The oligonucleotides can be administered, for example, orally, nasally, parenterally (intravenous, intramuscular, or subcutaneous), topically, transdermally, intravaginally, intravesically, intracistemally, or rectally. The dosage form can be, for example, a solid, semi-solid, lyophilized powder, or liquid dosage forms, such as for example, tablets, pills, soft elastic or hard gelatin capsules, powders, solutions, suspensions, suppositories, aerosols, or the like, for example, in unit dosage forms suitable for simple administration of precise dosages. A particular route of administration is oral, particularly one in which a convenient daily dosage regimen can be adjusted according to the degree of severity of the disease to be treated.

Auxiliary and adjuvant agents may include, for example, preserving, wetting, suspending, sweetening, flavoring, perfuming, emulsifying, and dispensing agents. Prevention of the action of microorganisms is generally provided by various antibacterial and antifungal agents, such as, parabens, chlorobutanol, phenol, sorbic acid, and the like. Isotonic agents, such as sugars, sodium chloride, and the like, may also be included. Prolonged absorption of an injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. The auxiliary agents also can include wetting agents, emulsifying agents, pH buffering agents, and antioxidants, such as, for example, citric acid, sorbitan monolaurate, triethanolamine oleate, butylated hydroxytoluene, and the like.

Solid dosage forms can be prepared with coatings and shells, such as enteric coatings and others well-known in the art. They can contain pacifying agents and can be of such composition that they release the active oligonucleotide or oligonucleotides in a certain part of the intestinal tract in a delayed manner. Examples of embedded compositions that can be used are polymeric substances and waxes. The active oligonucleotides also can be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. Such dosage forms are prepared, for example, by dissolving, dispersing, etc., the HDAC inhibitors or retinoic acid described herein, or a pharmaceutically acceptable salt thereof, and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol and the like; solubilizing agents and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol, dimethyl formamide; oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan; or mixtures of these substances, and the like, to thereby form a solution or suspension.

Generally, depending on the intended mode of administration, the pharmaceutically acceptable compositions will contain about 1% to about 99% by weight of the oligonucleotides described herein, or a pharmaceutically acceptable salt thereof, and 99% to 1% by weight of a pharmaceutically acceptable excipient. In one example, the composition will be between about 5% and about 75% by weight of a oligonucleotide described herein, or a pharmaceutically acceptable salt thereof, with the rest being suitable pharmaceutical excipients.

Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art, Reference is made, for example, to Remington's Pharmaceutical Sciences, 18th Ed. (Mack Publishing Company, Easton, Pa., 1990).

Kits

In other embodiments, kits are provided. Kits according to the disclosure include package(s) comprising oligonucleotides, peptides, peptide-oligonucleotide-conjugates, or compositions of the disclosure, In some embodiments, kits comprise a peptide-oligonucleotide-conjugate according to Formulae I, Ia, or Ib, or a pharmaceutically acceptable salt thereof. In other embodiments, kits comprise an oligonucleotide according to Formulae II or IIa, or a pharmaceutically acceptable salt thereof. In still other embodiments, kits comprise a peptide according to Formula III, or a pharmaceutically acceptable salt thereof.

The phrase “package” means any vessel containing oligonucleotides or compositions presented herein. In some embodiments, the package can be a box or wrapping. Packaging materials for use in packaging pharmaceutical products are well-known to those of skill in the art. Examples of pharmaceutical packaging materials include, but are not limited to, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment.

The kit can also contain items that are not contained within the package, but are attached to the outside of the package, for example, pipettes.

Kits can further contain instructions for administering oligonucleotides or compositions of the disclosure to a patient. Kits also can comprise instructions for approved uses of oligonucleotides herein by regulatory agencies, such as the United States Food and Drug Administration. Kits can also contain labeling or product inserts for the oligonucleotides. The package(s) or any product insert(s), or both, may themselves be approved by regulatory agencies. The kits can include oligonucleotides in the solid phase or in a liquid phase (such as buffers provided) in a package. The kits can also include buffers for preparing solutions for conducting the methods, and pipettes for transferring liquids from one container to another.

EXAMPLES

Examples have been set forth below for the purpose of illustration and to describe certain specific embodiments of the disclosure. However, the scope of the claims is not to be in any way limited by the examples set forth herein. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art and such changes and modifications including, without limitation, those relating to the chemical structures, substituents, derivatives, formulations or methods of the disclosure may be made without departing from the spirit of the disclosure and the scope of the appended claims. Definitions of the variables in the structures in the schemes herein are commensurate with those of corresponding positions in the formulae presented herein.

Example 1: General Method for Peptide Preparation and Purification Fast-Flow Peptide Synthesis

Peptides were synthesized on a 0.1-mmol scale using an automated flow peptide synthesizer. ChemMatrix Rink Amide HYR resin (200 mg) was loaded into a reactor maintained at 90° C. All reagents were flowed at 80 mL/min with HPLC pumps through a stainless steel loop maintained at 90° C. before introduction into the reactor. For each coupling, 10 mL of a solution containing 0.2 M amino acid and 0.2 M HAM in DMF were mixed with 200 μL diisopropylethylamine and delivered to the reactor. Fmoc removal was accomplished using 10.4 mL of 20% (v/v) piperidine. Between each step, 15 mL of DMF were used to wash out the reactor. The final coupling was with 4-pentynoic acid, rather than an amino acid, but using the same conditions. After completion of the synthesis, the resins were washed 3 times with DCM and dried under vacuum.

Peptide Cleavage and Deprotection

Each peptide was subjected to simultaneous global side-chain deprotection and cleavage from resin by treatment with 6 mL of Reagent K (82.5% trifluoroacetic acid, 5% phenol, 5% water, 5% thioanisole, and 2.5% 1,2-ethanedithiol (EDT)). Cleavages were left at room temperature for 16 hours to ensure complete removal of PH The cleavage cocktail was filtered to remove the resin and was evaporated by bubbling N2 through the mixture. Then ˜35 mL of cold ether was added and the crude product was pelleted through centrifugation for three minutes. This ether trituration and centrifugation was repeated two more times. After the third wash, the pellet was redissolved in 50% water and 50% acetonitrile and lyophilized

Peptide Purification

-   Solvent A: water containing 0.1% TFA -   Solvent B: acetonitrile containing 0.1% TFA

Lyophilized peptide was dissolved into a minimum volume of mobile phase (95% A, 5% B). The solution was loaded onto a reversed-phase HPLC column (Agilent Zorbax SB C18 column: 9.4×250 mm, 5 μm or Agilent Zorbax SB C3 column: 9.4×250 mm, 5 μm) attached to a mass-based purification system. A linear gradient was run at 0.5% B/min from 5% B to 55% B. Using mass data about each fraction from the instrument, only pure fractions were pooled and lyophilized. The purity of the fraction pool was confirmed by LC-MS.

Using the protocol of Example 1, the peptides of Table 2 were synthesized.

TABLE 2 Arginine-rich peptides for bicyclization and control sequences Peptide Sequence[a] Cyclic form 1 ZCRRRRRRCRRRRRRC Trithiol bicycle 2 ZCRRRCRRRC Trithiol bicycle 3 ZCRRRRRRCCRRRRRRC Double macrocycle 4 ZRRRRRRCCRRRRRR Macrocycle 5 ZRRRRRRRRRRRR None 6 ZCRRRRRRRRRRRRC Macrocycle [a]Z = 4-pentynoyl; R = arginine; C = cysteine; all peptides are C-terminal amides

Example 2: Synthesis of 1a and 2a

Peptides 1 (28 mg, 10 μmol) or 2 (20 mg, 12 μmol) were dissolved in DMF, and stock solutions of decafluorobiphenyl in DMF and diisopropylcthylamine (DIEA) were added for a final concentration in the reaction vessel of 1 mM peptide, 100 mM decafluorobiphenyl, and 50 mM DIEA. After two hours, DMF was removed by rotary evaporation to a final volume of 1 mL and the reaction was quenched by adding 39 mL of 85:15 water:acetonitrile containing 2% TFA. The crude reaction was centrifuged, filtered, and purified by mass-directed semi-preparative reversed-phase HPLC as described above to provide 1a (13 mg, 35%) or 2a (11 mg, 35%).

Example 3: Synthesis of 1b and 2b

Purified peptides 1a (10 mg, 2.56 μmol) or 2a (5 mg, 1.9 μmol) were dissolved in DMF, and stock solutions of 1,3,5-benzenetrithiol in DMF and DIEA were added such that the final concentration in the reaction vessel was 1 mM peptide, 1 mM 1,3,5-benzenetrithiol, and 50 mM DIEA. The crude reaction was purified by mass-directed semi-preparative reversed phase HPLC (Agilent Zorbax SB C3 column: 9.4×250 mm, 5 μm). After two hours, DMF was removed by rotary evaporation to a final volume of 1 mL and the reaction was quenched by adding 39 mL of 85:15 water:acetonitrile containing 2% TFA. The crude reaction was purified by mass-directed semi-preparative reversed-phase HPLC as described above to provide 1b (6.4 mg, 62%) or 2b (1.8 mg, 35%).

Example 4: Synthesis of 3b Using Orthogonal Deprotection

Peptide 3 was synthesized using standard automated flow chemistry except tert-butyl thiol protected cysteine was incorporated for cysteine residues 1 and 2 and trityl protected cysteine was incorporated for cysteine residues 3 and 4. After cleavage and deprotection, the peptide was purified by reversed-phase HPLC. The peptide was macrocyclized using a procedure analogous to Example 3. tert-butylthiol protected cysteine residues 1 and 2 were deprotected by incubating the peptide (1 mM) with DTT (50 mM) and Tris (25 mM pH 8) in water. After solid-phase extraction (SPE) to remove residual DTT, the remaining two free cysteine residues were macrocyclized using a procedure analogous to Example 3.

Example 5: Synthesis of 3b Using Controlled Bicyclization

Procedure for Synthesis of3a

Peptide 3 was synthesized using standard automated flow chemistry using 200 mg of resin except tort-butyl thiol protected cysteine was incorporated for cysteine residues 1 and 3 and trityl protected cysteine was incorporated for cysteine residues 2 and 4. The tert-butyl thiol groups were deprotected on resin using 3.8 mmol DTT in 2.5 rnL DMF with 0.25 mL DIEA. The reaction proceeded at 60° C. for 20 minutes. The resin was washed 3 times with DMF, followed by the addition of decafluorobiphenyl (1 mmol) with 2.5 mL DMF and 0.25 mL DIEA. The reaction proceeded at room temperature for 2 hours, and then the resin was washed with DMF (3×) and DCM (3×). The peptide was then cleaved from the resin and purified as normal to afford 3a.

Procedure for Synthesis of Bicycle 3b Controlled Bicyclization

Peptide 3a (15 mg, 4 μmol, assuming 6 coordinated TFA salts) was suspended in 4 mL DMG. Neat DIEA (35.4 μL) was added to achieve a concentration of 50 mM. The reaction was allowed to proceed for 5 minutes, after which complete conversion is observed. DMF was removed by rotary evaporation to a final volume of 1 mL and the reaction was quenched by adding 39 mL of water containing 2% TFA, The reaction was purified by mass-directed semi-preparative reversed-phase HPLC to provide peptide 3b (12.0 mg, 81% yield).

Example 6: Synthesis of 6c

Peptide 6 was synthesized using standard automated flow chemistry using 200 mg of resin except tert-butyl thiol protected cysteine was incorporated for cysteine residue 1 and trityl protected cysteine was incorporated for cysteine residue 2. The teri-butyl groups were deprotected on resin using 3.8 mmol DTT in 2.5 mL DMF with 0.25 mL DIEA. The reaction proceeded at 60° C. for 20 minutes. The resin was washed 3× with DMF, followed by the addition of decafluorobiphenyl (1 mmol) with 2.5 mL DMF and 0.25 mL DIEA. The reaction proceeded at room temperature for 2 hours, and then the resin was washed with DMF (3×) and DCM (3×). The peptide was then cleaved from the resin and purified as normal to afford the monocryl 6a.

Peptide 6a was macrocyclized by suspending the peptide (12 mg, 3.9 μmol, assuming 6 coordinated TFA salts) was suspended in 3.9 mL DMF. Neat DIEA (33.8 μL) was added to achieve a concentration of 50 mM. The reaction was allowed to proceed for 5 minutes, after which complete conversion was observed. DMF was removed by rotary evaporation to a final volume of 1 mL and the reaction was quenched by adding 39 mL of water containing 2% TFA. The reaction was purified by mass-directed semi-preparative reversed-phase HPLC to provide peptide 6c (8.4 mg, 88% yield).

Example 6: Peptide Conjugation

Procedure for Coupling 5-azidopentanoic to PMO

PMO IVS-654 (R₂=5′-GCT ATT ACC TTA ACC CAG-3′; z=18) (200 mg, 32 μmol) was dissolved in 600 μL DMSO. To the solution was added a solution containing 4 equivalents of 5-azidopentanoic acid (13.6 μL, 128 μmol) activated with HBTU (320 μL of 0.4 M HBTU DMF, 128 μmol) and DIEA (22.3 μL, 128 μmol) in 244 μL DMF (Final reaction volume=1.2 mL). The reaction proceeded for 25 minutes before being quenched with 1 mL of water and 2 mL of ammonium hydroxide. The ammonium hydroxide will hydrolyze any ester formed during the course of the reaction. After 1 hour, the solution was diluted to 40 mL and purified using reversed-phase HPLC (Agilent Zorbax SB C3 column: 21.2×100 mm, 5 μm) and a linear gradient from 2 to 60% B (solvent A: water; solvent B: acetonitrile) over 58 minutes (1% B min). Using mass data about each fraction from the instrument, only pure fractions were pooled and lyophilized. The purity of the fraction pool was confirmed by LC-MS. Lyophilization afforded 171 mg of dry powder (84% yield).

General Procedure for PMO-Peptide Conjugation by Azide/Alkyne Huisgen Cycloaddition

A 20 mL scintillation vial with a septum cap was charged with peptide alkyne (1.1 μmol), ISV2-654 azide (0.95 μmol), and copper bromide (0.05 mmol). The vial was purged with nitrogen for 5 minutes to ensure the removal of oxygen before the addition of ˜1 mL of DMF through the septum. The reaction mixture was vortexed for 1 minute. After 2 hours, the reaction mixture was diluted with 10 mL of 50 mM Iris (pH 8), and loaded onto reversed-phase HPLC column (Agilent Zorbax SB C3 9.4×50 mm, 5 μm). Chromatography was performed using a linear gradient from 5-45% B over 20 minutes. Solvent A: 5 mM ammonium acetate, pH=8 in water; solvent B: 5 mM ammonium acetate pH=8 in 90% acetonitrile 10% water, Using mass data about each fraction from the instrument, only pure fractions were pooled and lyophilized. The purity of the fraction pool was confirmed by LC-MS.

Example 7: Proteolysis Assay

For each peptide, 19.6 μL of PBS, 0.2 μL of Trypsin (0.005 mg/mL stock solution in 1 mM HCl), and 0.2 μL of peptide (1 mM DMSO stock solution) were combined in a PCR tube. The resulting reaction mixture was capped and incubated at 37° C. At each time point, 1.0 μL of the crude reaction was transferred to a LC-MS vial and quenched by addition of 99 μL of 50:50 water:acetonitrile containing 0.1% TFA. 1.0 μL of the quenched reaction was injected onto the Agilent 6550 iFunnel Q-TOF MS. Time points were taken at t=0 min, 20 min, 40 min, and 60 min. An extracted ion current (EIC) for the +5 charge state m/z was analyzed using the MassHunter software. The EIC peak was integrated and percent peptide intact was determined by (EICt₁/EICt₀)*100 in which EICt₁ is the peak integration at a given time point and EICt₀ is the peak integration at time t=0. Results are shown in FIG. 2.

Example 8: In Vivo Administration of Peptide-PMO Conjugates in EGFP-654 Mice

A eGFP-based assay for in vivo antisense activity was used to evaluate oligomers comprising modified intersubunit linkages. The transgenic eGFP mouse model in which the eGFP-654 transgene is expressed uniformly throughout the body has been described previously. This model uses a splicing assay for activity in which the modified oligomers of the present invention block aberrant splicing and restore correct splicing of the modified enhanced green fluorescent protein (eGFP) pre-mRNA. In this approach, antisense activity of each oligomer is directly proportional to up-regulation of the eGFP reporter. As a result, the functional effects of the same oligomer can be monitored in almost every tissue, This is in contrast to oligomers targeted to genes whose expression is restricted to or is phenotypically relevant in only certain tissues. In the eGFP-654 mice, the pre-mRNA was readily detectable in all tissues, although smaller amounts were found in the bone marrow, skin, and brain. The level of translated eGFP is proportional to the potency of the antisense oligomers and their concentration at the site of action. RT-PCR of total RNA isolated from various tissues showed expression of eGFP-654 transcript in all tissues surveyed.

The specific peptide-PMO conjugate described in this example are the cyclic and bicyclic peptides coupled to PMO-IVS₂-654 (described in Example 6).

These peptide-PMO conjugates, targeting the eGFP transgene were administered into the left lateral ventricle of EGFP-654 mice by a single intracebreroventricular (ICV) injection using a tereotaxis apparatus. Doses consisted of either 5 mg/kg for all mice or spanned a range of doses. Two weeks post-injection, mice were euthanized and the brain was removed and cut in half sagittally at the midline into left and right hemispheres. Each hemisphere was imaged on a Typhoon Trio (GE) by placing the cut surface face down on the flatbed platen. Scans were collected using a 488 nm laser to excite eGFP fluorescence. The resulting images were analyzed with ImageQuant software (GE) to quantify the fluorescence intensity of each hemisphere. The total detected fluorescence intensity within each hemisphere was divided by the number of pixels present in that hemisphere to yield an area-independent average fluorescence value for each half of the brain. Activity results for each surviving animal in a treatment group are expressed as points on the scatter plot. The mean fluorescence of the group is indicated by the horizontal line, +/−SD.

FIG. 1 shows that cyclic and bicyclic peptides coupled to PMO-IVS₂-654 (described in Example 6) demonstrate increased activity in a cellular exon-skipping assay. IVS₂-654 corrects splicing of a mutated eGFP pre-mRNA. An increase in splice correction leads to an increase in fluorescence. Results have been normalized to the unconjugated IVS₂-654 PMO.

INCORPORATION BY REFERENCE

The contents of all references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated herein in their entireties. Unless otherwise defined, all technical and scientific terms used herein are accorded the meaning commonly known to one with ordinary skill in the art.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the following claims. 

What is claimed is:
 1. A peptide-oligonucleotide conjugate of Formula I:

or a pharmaceutically acceptable salt thereof, wherein: A′ is selected from —NHCH₂C(O)NH₂, —N(C₁₋₆-alkyl)CH₂C(O)NH₂,

wherein R⁵ is —C(O)(O-alkyl)_(x)-OH, wherein x is 3-10 and each alkyl group is independently at each occurrence C₂₋₆-alkyl, or R⁵ is selected from —C(O)C₁₋₆-alkyl, trityl, monomethoxytrityl, -(C₁₋₆-alkyl)R⁶, -(C₁₋₆-heteroalkyl)-R⁶, aryl-R⁶, heteroaryl-R⁶, —C(O)O-C₁₋₆-alkyl)-R⁶, —C(O)O-aryl-R⁶, —C(O)O-heteroaryl-R⁶, and

wherein R⁶ is selected from OH, SH, and NH₂, or R⁶ is O, S, or NH, covalently linked to a solid support; each R¹ is independently selected from OH and —NR³R⁴, wherein each R³ and R⁴ are independently at each occurrence -₁₋₆-alkyl; each R² is independently selected from H, a nucleobase, and a nucleobase functionalized with a chemical protecting-group, wherein the nucleobase independently at each occurrence comprises a C₃₋₆-heterocyclic ring selected from pyridine, pyrimidine, triazinane, purine, and deaza-purine; z is 8-40; and E′ is selected from H, -C₁₋₆-alkyl, —C(O)C₁₋₆-alkyl, benzoyl, stearoyl, trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl,

wherein Q is —C(O)(CH₂)₆C(O)— or —C(O)(CH₂)₂S₂(CH₂)₂C(O)—, and R⁷ is —(CH₂)₂OC(O)N(R⁸)₂, wherein R⁸ is —(CH₂)₆NHC(═NH)NH₂, and wherein L is covalently linked by an amide bond to the amino-terminus of J, and L is —C(O)(CH₂)₁₋₆-C₁₋₆-heteroaromatic-(CH₂)₁₋₆C(O); t is 4-16; each J is independently at each occurrence selected from an amino acid of the structure

wherein: r and q are each independently 0, 1, 2, 3, or 4; and each R⁹ is independently at each occurrence selected from H, an amino acid side-chain, and an amino acid side-chain functionalized with a protecting-group, wherein three or four amino acid side-chains of R⁹ independently at each occurrence comprise a sulfur atom: a) wherein when the number of sulfur atoms is three, then the sulfur atoms, together with the atoms to which they are attached, form the structure

such that the amino acids form a bicyclic structure; or b) wherein when the number of sulfur atoms is four, then the sulfur atoms, together with the atoms to which they are attached, form two occurrences of the structure

such that the amino acids form a bicyclic structure; wherein d is 0 or 1, and M is selected from:

wherein each R¹⁰ is independently at each occurrence H or a halogen; and G is covalently linked by an amide bond to the carboxy-terminus of J, and G is selected from H, —C(O)C₁₋₆-alkyl, benzoyl, and stearoyl, and wherein at least one of the following conditions is true: 1) A′ is

or 2) E′ is


2. The peptide-oligonucleotide conjugate of claim 1, or a pharmaceutically acceptable salt thereof; wherein A′ is selected from —N(C₁₋₆-alkyl)CH₂C(O)NH₂,


3. The peptide-oligonucleotide conjugate of any one of claims 1-2, or a pharmaceutically acceptable salt thereof; wherein E′ is selected from. H, —C(O)CH₃, benzoyl, stearoyl, trityl, 4-methoxytrityl, and


4. The peptide-oligonucleotide conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the peptide-oligonucleotide conjugate of Formula I is a peptide-oligonucleotide conjugate selected from:

wherein E′ is selected from H, C₁₋₆-alkyl, —C(O)CH₃, benzoyl, and stearoyl.
 5. The peptide-oligonucleotide conjugate of claim 1 or 4, or a pharmaceutically acceptable salt thereof, wherein the peptide-oligonucleotide conjugate is of the formula (Ia).
 6. The peptide-oligonucleotide conjugate of claim 1 or 4, or a pharmaceutically acceptable salt thereof, wherein the peptide-oligonucleotide conjugate is of the formula (Ib).
 7. The peptide-oligonucleotide conjugate of any one of claims 1-6, or a pharmaceutically acceptable salt thereof, wherein each R¹⁰ is fluorine.
 8. The peptide-oligonucleotide conjugate of any one of claims 1-7, or a pharmaceutically acceptable salt thereof, wherein M is


9. The peptide-oligonucleotide conjugate of any one of claims 1-8, or a pharmaceutically acceptable salt thereof, wherein each J is independently selected from cysteine and arginine.
 10. The peptide-oligonucleotide conjugate of any one of claims 1-9, or a pharmaceutically acceptable salt thereof, wherein three J groups are cysteine.
 11. The peptide-oligonucleotide conjugate of any one of claims 1-10, or a pharmaceutically acceptable salt thereof, wherein four J groups are cysteine.
 12. The peptide-oligonucleotide conjugate of any one of claims 1-11, or a pharmaceutically acceptable salt thereof, wherein each R¹ is N(CH₃)₂.
 13. The peptide-oligonucleotide conjugate of any one of claims 1-12, or a pharmaceutically acceptable salt thereof, wherein each R² is a nucleobase independently at each occurrence selected from adenine, guanine, cytosine, 5-methyl-cytosine, thymine, uracil, and hypoxanthine.
 14. The peptide-oligonucleotide conjugate of any one of claims 1-13, or a pharmaceutically acceptable salt thereof, wherein L is —C(O)(CH₂)₁₋₆-triazole-(CH₂ ₁₋₆C(O)—.
 15. The peptide-oligonucleotide conjugate of any one of claims 1-14, or a pharmaceutically acceptable salt thereof, wherein L is


16. The peptide-oligonucleotide conjugate of any one of claims 1-15, or a pharmaceutically acceptable salt thereof, wherein G is selected from H, C(O)CH₃, benzoyl, and stearoyl.
 17. The peptide-oligonucleotide conjugate of any one of claims 1-16, or a pharmaceutically acceptable salt thereof, wherein G is H. 18, The peptide-oligonucleotide conjugate of claim 1-17, or a pharmaceutically acceptable salt thereof, wherein d is
 1. 19. The peptide-oligonucleotide conjugate of any one of claims 1-18, or a pharmaceutically acceptable salt thereof, wherein d is
 0. 20. The peptide-oligonucleotide conjugate of any one of claims 1-19, or a pharmaceutically acceptable salt thereof, wherein

is selected from:

wherein R is arginine and C is cysteine; and G is selected from H, C(O)CH₃, benzoyl, and stearoyl, wherein G is covalently linked by an amide bond to the carboxy terminus of the peptide.
 21. The peptide-oligonucleotide conjugate of any one of claims 1-18 and 20, or a pharmaceutically acceptable salt thereof, wherein the peptide-oligonucleotide conjugate is selected from:

wherein G H or —C(O)CH₃, wherein G is covalently attached by an amide bond to the carboxy terminus of the peptide; C is cysteine; R is arginine; R² is a nucleobase, independently at each occurrence, selected from adenine, guanine, cytosine, 5-methyl-cytosine, thymine, uracil, and hypoxanthine; and z is 8-40.
 22. A composition comprising a compound of any one of claims 1-21, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier.
 23. A method of treating a muscle disease, a viral infection, or a bacterial infection in a subject in need thereof, comprising administering to the subject a compound of any one of claims 1-21 or a composition of claim
 22. 24. The method according to claim 23, wherein the muscle disease is Duchenne Muscular Dystrophy.
 25. The method according to claim 23, wherein the viral infection is caused by a virus selected from the group consisting of marburg virus, ebola virus, influenza virus, and dengue virus. 